Pancreatic ductal adenocarcinoma (PDA) is a highly lethal human malignancy, typically diagnosed at an advanced stage and known to be largely unresponsive to chemotherapy and ionizing radiation. Recent genomic characterization of PDA reveals that between 20-25 % of PDA harbor recurrent mutations in genes, including BRCA1/2, PALB2, and ATM, which are critical for homologous recombination (HR), an important form of DNA repair. In many patients, these may be germline mutations. This subgroup of PDAs, termed HR-deficient PDA, has emerged as a defined biological entity associated with increased chemoresistance and a more aggressive disease course. The defects in HR observed in these tumors impart cells with a specific vulnerability to PARP inhibitors and platinum-containing therapy. Still, as observed in the case of many other targeted therapies, only a fraction of HR-defective patient tumors respond to PARP inhibition. More so, many patients that initially respond eventually often develop resistance and progress. Therefore, novel therapies which can be effective against HR- defective PDA, alone or in combination with PARP inhibitors or other combinatorial regimens, are urgently needed. We have recently determined that inactivation of the HR pathway is associated with overexpression of polymerase theta (Pol?, also known as POLQ) in PDA. POLQ is a key enzyme that regulates an alternative pathway of DNA repair, known as the alternative non-homologous end-joining (Alt-NHEJ) pathway. Data from our group indicates that in the setting of defective HR, Alt-NHEJ becomes a critical pathway responsible for the repair of DNA breaks. Furthermore, we show that POLQ inhibition in HR-defective tumor cells demonstrates a synthetic lethality phenotype, not observed in cells with intact HR. In this proposal, we present exciting new data that knockdown of POLQ is synthetically lethal in PDA cells deficient for Brca1, Brca2, Atm, and Palb2 genes. POLQ knockdown significantly inhibited growth of both Brca2- and Atm-deficient tumors cells in vivo. Further, POLQ knockdown significantly upregulated the cGAS-STING pathway in HR-deficient PDA and promoted T cell infiltration. Here, we plan to examine the unique role of POLQ in pancreatic cancer biology and its role as a novel therapeutic target in HR-defective pancreatic cancers. We will also evaluate the antitumor effect of combining POLQ inhibition with: i) current standard cytotoxic chemotherapies, ii) PARP inhibition, and iii) immunotherapy. An important goal of this proposal is to generate a set of data for proof-of-concept that targeting POLQ in a valuable therapeutic strategy in HR-defective pancreatic cancer, as POLQ inhibitors are currently in development for clinical use.